The prominent mitochondrial protein kinases (mPKs) comprising pyruvate dehydrogenase kinases (PDKs), and branched-chain !-ketoacid dehydrogenase kinase (BCK) are the molecular switches that control carbohydrate and branched-chain amino acid degradation. Mitochondrial PDKs (isoforms 1, 2, 3 and 4) are tethered to the E2p/E3BP core of the mammalian pyruvate dehydrogenase complex (PDC). These mPKs down-regulate activity of the mitochondrial PDC by reversible phosphorylation, in response to hormonal and nutritional stimuli. Certain PDK isoforms are over-expressed in disease states such as type 2 diabetes, obesity and cancer, resulting in impaired glucose oxidation. Toward understanding the structure and function of these PDKs, The P.I.[unreadable]s laboratory has determined the crystal structures for isoforms 1, 2, and 3, and various PDKinhibitor/ activator complexes. Based on these advances, the P.I. proposes to continue our investigation into the structure, function and regulation of mammalian PDKs. The Specific Aims are: 1) To dissect the allosteric mechanisms by which the L2 domain and synthetic ligands modulate PDK3 and PDK1 activities; 2) To offer biochemical and structural basis for the hyper-reactivity of PDK4 and to identify the E1p substrate-binding site in this kinase isoform; 3) To decipher the molecular mechanisms underlying the inactivation of PDC by PDKmediated phosphorylation of the E1p substrate. Standard methods including X-ray crystallography and isothermal titration calorimetry and kinase activity assays that have been established in this laboratory will be employed to achieve these Specific Aims. Understanding the structural and biochemical mechanisms for these PDK isoforms will provide a paradigm for developing new PDK inhibitors to ameliorate human disease caused by aberrant glucose catabolism.